Germinal centers (GCs) are of tremendous importance to human health. Not only do they generate the high-affinity antibodies that make vaccination possible and are the cause of allergies and humoral autoimmune diseases, but they are also the source of the genetic lesions that trigger most B cell malignancies. There is a fundamental gap in our understanding of how the selection of B cell clones during the GC reaction can generate the highly mutated antibodies necessary, for example, for broad neutralization of HIV. This gap represents a major obstacle to the development of effective vaccines for many human diseases. The long-term goal of the investigator's research is to develop a detailed understanding, at the cellular and molecular levels, of all aspects of the B cel response, from first contact with antigen to production of high-affinity antibodies. Recent work by the investigator and others has underscored the importance of interaction between T follicular helper (Tfh) cells and B cells in GC selection. The objective of this application is to investigate specific aspects of this interaction, especially with regard to its influence on B cell fate choice. The rationale for the proposed research is that, because Tfh cells control key aspects of the GC-such as magnitude, persistence, and selective pressure-understanding the relationship between B cells and Tfh cells should improve our ability to coax GCs into producing the highly mutated antibodies required for broad neutralization. In this context, three specific aims are proposed. First, in vivo photoactivation by multiphoton microscopy, a method developed by the investigator, will be used to determine the role of accessibility of GCs to incoming Tfh cells in determining how T cell specificity in the GC is controlled. The hypothesis in this aim is that GCs are open to the ingress and egress of Tfh cells, and that this openness impacts the clonality and specificity of GC T cells and the longevity of GC reactions. Second, a method to trigger selection of GC B cells by Tfh cells in vivo, also developed by the investigator, will be used to probe the gene expression changes that are induced in B cells upon selection. The hypothesis in this aim is that Tfh cell help triggers specific gene expression programs that determine the subsequent fate choices made by of GC B cells. Finally, the investigator proposes to develop a novel approach to measuring the history of interactions between Tfh cells and B cells in vivo. This technique will allow for interactions between immune cells to be probed and quantified in a technically much simpler manner, and in a more physiological context, than by currently available methods. The proposed research is significant because it will contribute to our understanding of key aspects of GC selection at the cellular and molecular levels, especially with regard to its reliance on T cell help. This knowledge should allow better control over the GC reaction, especially with regard to its longevity and output. Ability to control these aspects of te GC is a prerequisite for achieving broad neutralization by vaccination.